In recent years, a nitrile hydratase was found, which is an enzyme having nitrile hydrolysis activity that catalyses the hydration of a nitrile group to its corresponding amide group. Also, methods are disclosed to produce corresponding amide compounds from nitrile compounds using the enzyme or a microbial cell or the like containing the enzyme. Compared with conventional chemical synthetic methods, such methods are known by a high conversion rate or selectivity rate from a nitrile compound to a corresponding amide compound.
Examples of microorganisms that produce a nitrite hydratase are the genus Corynebacterium, genus Pseudomonas, genus Rhodococcus, genus Rhizobium, genus Klebsiella, genus Pseudonocardia and the like. Among those, Rhodococcus rhodochrous J1 strain has been used for industrial production of acrylamides, and its usefulness has been verified. Furthermore, a gene encoding a nitrile hydratase produced by the J1 strain has been identified (see patent publication 1).
Meanwhile, introduction of a mutation into a nitrile hydratase has been attempted not only as a way to use a nitrile hydratase isolated from naturally existing microorganisms or the gene of such a nitrile hydratase, but also as a way to change the activity, substrate specificity, Vmax, Km, heat stability, stability in a substrate, stability in a subsequent product and the like of a nitrile hydratase (see patent publication 2). Nitrile hydratase genes with improved heat resistance and amide-compound resistance have been produced by the inventors of the present invention (patent publications 3 and 4).
However, considering production costs such as the cost of catalysts when producing amide compounds, it is useful to develop a nitrile hydratase with further improved heat resistance and amide-compound resistance, and enhanced capability of reacting at a high temperature. Thus, obtaining enzymes with such improved properties is highly desired for the purpose of reducing the amount of enzymes needed for reactions, lowering production costs and the like.